I405V and TaqIB polymorphisms of the cholesteryl ester transfer protein and their relation to serum lipid and lipoprotein levels in a Turkish population

Cholesteryl ester transfer protein (CETP) plays a central role in high‐density lipoprotein (HDL) metabolism. Genetic polymorphisms of the CETP gene can influence levels of serum lipoproteins. It has been reported that mean HDL‐cholesterol (HDL‐C) concentrations are low in Turkish population. Thus, we investigated the frequencies of the common I405V and TaqIB polymorphisms of the CETP gene and their relation to serum lipid and lipoprotein levels in a Turkish population. The variant allele frequencies of I405V and TaqIB polymorphisms of the CETP gene were found to be 0.38 and 0.46, respectively and similar to some of the European populations. Subjects for the VV genotype of I405V polymorphism had higher HDL‐C levels than did II subjects. The covariance analysis showed that gender and triglyceride (TG) levels have an effect on the association of HDL‐C and I405V polymorphism. In conclusion, our results indicate that I405V polymorphism may affect the HDL‐C levels in Turkish population. The association of this polymorphism and HDL‐C levels could be modified by other factors, such as gender and TG levels. Copyright © 2009 John Wiley & Sons, Ltd.     

A Novel Missense Mutation （L^296Q） in Cholesteryl Ester Transfer Protein Gene Related to Coronary Heart Disease

Cholesteryl ester transfer protein (CETP) is a key participant in the reverse transport of cholesterol from the peripheral tissues to the liver. To understand the role that CETP gene plays in the pathogenesis of coronary heart disease (CHD) , the promoter region, all 16 exons and adjacent intronic regions of CETP gene were screened for single nucleotide polymorphisms (SNPs) in 203 CHD patients and 209 controls by a combination of PCR, denaturing high performance liquid chromatography (DHPLC), molecular cloning, and DNA sequencing. A novel missense mutation in the CETP gene was identified. This mutation (L296Q) was a T-to-A conversion at codon 296 of exon 10 which replaced the codon for leucine (CTG) with the codon for glutamine (CAG). Association study revealed that L296Q mutation was associated with CHD with a significantly higher mutant allele frequency in the CHD patients than that in the controls (0. 160 vs. 0.091,x2= 9.014, P = 0.003), and that the odds ratio for the development of CHD was 1.83 for     

Penetration of an emulsion surface by cholesteryl ester transfer protein

Quenching of the intrinsic fluorescence of cholesteryl ester transfer protein (CETP) by spin labelled fatty acids (5-NS and 16-NS) was investigated to determine the degree to which the protein penetrated the phospholipid monolayer surface of a lipid emulsion. When bound to the phospholipid surface approximately 50% of the fluorophores of the transfer protein were accessible to quenching by 5-NS whose nitroxy group locates near the monolayer surface. On the other hand, only 22% of the fluorophores of CETP were accessible to quenching by 16-NS whose nitroxy group locates deeper in the surface monolayer. Quenching of the CETP fluorescence by an aqueous phase quencher (acrylamide) shows that the protein undergoes a conformational change on binding which increases the proportion of the tryptophan residues exposed to the aqueous phase. The results indicate that CETP does not penetrate the lipid surface to a significant degree. Received: 29 March 1996 / Accepted: 30 May 1996     

Genetic variants affecting alternative splicing of human cholesteryl ester transfer protein

Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (Δ9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T > C located in intron 8 in a putative splicing branch site and rs5883 C > T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that Δ9-CETP protein is expressed in the liver but fails to circulate in the blood.     

Binding profiles of cholesterol ester transfer protein with current inhibitors: a look at mechanism and drawback

Although the pharmacological inhibition of cholesterol ester transport protein (CETP) has been proposed as a method of preventing and treating cardiovascular disease (CVD), the adverse effects of current inhibitors have cast doubt on the interaction mechanisms of inhibitors and CETP. In response, a molecular dynamics simulation was used to investigate their interaction and shed light on the lipid exchange mechanism of CETP. Results showed that torcetrapib, anacetrapib, and evacetrapib can induce the incremental rigidity of CETP, yet decrease the stability of Helix X and the hydrophobic tunnel of CETP, with passable binding abilities (ΔG_bind, ?61.08, ?64.23, and ?61.57 kcal mol^?1). During their binding processes, Van der Waals components (ΔE_vdw + ΔG_SA) play a dominant role, and the inhibitory effects closely correlated with residues Cys13, Val198, Gln199, Ser230, His232, and Phe263, which could reduce the flexibility of N- and C- termini and Helix X, as well as the stability of hydrophobic tunnel, into which the three inhibitors could enter and promote the formation of intramolecular H-bonds such as Thr138–Asn192 and Arg37–Glu186. Additionally, the three inhibitors could restrain the formation of an opening at the CETP N-terminal, which given the other findings suggests the tunneling mechanism of CETP transfer. The paper closes with an explanation of conceivable causes of the insufficient efficacy of the inhibitors, and puts forward the rationality in targeting the CETP distal end for CVD therapies.     

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic,CETP transgenic,LDLr-/- mice

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.     

The effect of dietary copper on rat plasma apolipoprotein B,E plasma levels,and apolipoprotein gene expression in liver and intestine

The plasma levels of apo B and apo E, and the level of hepatic and intestinal mRNA coding for these apolipoproteins were investigated in weanling male rats pair-fed for 6 wk with a control or copperdeficient diet. Plasma cholesterol, triglycerides, and phospholipids were significantly increased, and plasma apo B and apo E levels were also markedly increased in copper-deficient rats as compared to control rats. Copper deficiency significantly increased triglyceride levels and decreased cholesterol levels in the liver. No major differences in the levels of hepatic and intestinal apo B and apo E mRNA occurred between control and copper-deficient rats. These data imply that hypertriglyceridemia dn hypercholesterolemia owing to the copper deficiency are not accompanied by modifications in the gene expression at the mRNA level in the liver and intestine of the apolipoproteins studied.     

Apolipoprotein E gene polymorphism,hypercholesterolemia and glomerular filtration rate in type 2 diabetic subjects: A 9-year follow-up study

OBJECTIVE: To study the association between apolipoprotein E (apoE) genotype and the rate of decline in glomerular filtration rate (GFR) in type 2 diabetic patients in a 9-year prospective study. METHODS: GFR was determined in 84 type 2 diabetic patients by plasma clearance of (51)Cr-EDTA at baseline and after 9 years of follow-up. ApoE genotypes were determined by polymerase chain reaction and restriction enzyme HHAI digestion and designated as epsilon4 allele group (apoE4/2, 4/3 and 4/4 genotypes; n = 20) and non-epsilon4 allele group (apoE3/3 and E3/2 genotypes; n = 64). We focused our analysis on those patients who were more likely to progress to diabetic renal disease, i.e. whose GFR fell more than expected in the normal course of ageing [1 ml x min(-1) x (1.73 m(2))(-1) per year]. RESULTS: In the whole population, the decline in the GFR did not differ statistically significantly between the apoE genotype groups [p = 0.65 with analysis of variance for repeated variables (RANOVA) for interaction between apoE genotype group and time point]. However, among patients whose GFR changed more than 9 ml x min(-1) x (1.73 m(2))(-1), GFR showed a statistically significantly greater decline in the epsilon4 allele group (n = 11) than in the non-epsilon4 allele group (n = 43) [from 116 +/- 36 to 80 +/- 29 ml x min(-1) x (1.73 m(2))(-1) vs. from 119 +/- 20 to 96 +/- 18 ml x min(-1) x (1.73 m(2))(-1); p = 0.005 with RANOVA]. CONCLUSION: ApoE allele epsilon4 may speed up the rate of decline of the GFR in patients with progressive diabetic renal disease.     

Effects of cholesterol ester transfer protein Taq1B gene polymorphism on serum lipoprotein levels in Turkish coronary artery disease patients

To evaluate the effect of cholesterol ester transfer protein (CETP) Taq1B gene polymorphism on serum lipid profile in Turkish coronary artery disease (CAD) patients, we investigated Taq1B gene polymorphism of CETP and serum lipid levels in 111 controls and in 173 CAD patients with myocardial infarction. There were no significant differences in the allele distribution at this polymorphic locus between the population sample and patients with coronary artery disease with myocardial infarction. To detect the association between the Taq1B RFLP and serum lipid levels, we determined the serum concentrations of total cholesterol, triglycerides and high density lipoprotein cholesterol (HDL-C) in the subjects studied and correlated the results to the Taq1B RFLP. Patients with Taq B1B1 genotypes had lower HDL-C levels than patients with B2B2 genotype (p = 0.003). Also in control subjects with Taq B1B1 genotype, lower HDL-C levels (p = 0.05) and higher triglyceride levels (p = 0.017) and body mass index (p = 0.05) were observed compared with control subjects with the B1B2 genotype. It was observed that in our population the distribution of CETP Taq1B genotypes is similar to other populations (except Greeks). The present study demonstrates that CETP Taq1B gene polymorphism may be responsible for low HDL cholesterol levels in patients with CAD and in healthy controls in Turkey.     

Conversion of lipid transfer inhibitor protein (apolipoprotein F) to its active form depends on LDL composition

Lipid transfer inhibitor protein (LTIP) exists in both active and inactive forms. Incubation (37°C) of plasma causes LTIP to transfer from a 470 kDa inactive complex to LDL where it is active. Here, we investigate the mechanisms underlying this movement. Inhibiting LCAT or cholesteryl ester transfer protein (CETP) reduced incubation-induced LTIP translocation by 40-50%. Blocking both LCAT and CETP completely prevented LTIP movement. Under appropriate conditions, either factor alone could drive maximum LTIP transfer to LDL. These data suggest that chemical modification of LDL, the 470 kDa complex, or both facilitate LTIP movement. To test this, LDL and the 470 kDa fraction were separately premodified by CETP and/or LCAT activity. Modification of the 470 kDa fraction had no effect on subsequent LTIP movement to native LDL. Premodification of LDL, however, induced spontaneous LTIP movement from the native 470 kDa particle to LDL. This transfer depended on the extent of LDL modification and correlated negatively with changes in the LDL phospholipid + cholesterol-to-cholesteryl ester + triglyceride ratio. We conclude that LTIP translocation is dependent on LDL lipid composition, not on its release from the inactive complex. Compositional changes that reduce the surface-to-core lipid ratio of LDL promote LTIP binding and activation.     

Constitutive inhibition of plasma CETP by apolipoprotein C1 is blunted in dyslipidemic patients with coronary artery disease

Plasma cholesteryl ester transfer protein (CETP) promotes the cholesterol enrichment of apoB-containing lipoproteins (VLDL and LDL) at the expense of HDL. Recent studies demonstrated that apoC1 is a potent CETP inhibitor in plasma of healthy, normolipidemic subjects. Our goal was to establish whether the modulation of CETP activity by apoC1 is influenced by dyslipidemia in patients with documented coronary artery disease (CAD). In the total CAD population studied (n = 240), apoC1 levels correlated negatively with CETP activity, independently of apoE-epsilon, CETP-Taq1B, and apoC1-Hpa1 genotypes. In multivariate analysis, the negative relationship was observed only in normolipidemic patients, not in those with hypercholesterolemia, hypertriglyceridemia, or combined hyperlipidemia. In the normolipidemic subjects, apoC1 levels were positively associated with higher HDL- to LDL-cholesterol ratio (r = 0.359, P < 0.001). It is concluded that apoC1 as a CETP inhibitor no longer operates on cholesterol redistribution in high-risk patients with dyslipidemia, probably due to increasing amounts of VLDL-bound apoC1, which is inactive as a CETP inhibitor. Patients with dyslipidemia could experience major benefits from treatment with pharmacological CETP inhibitors, which might compensate for blunted endogenous inhibition.     

载脂蛋白E基因多态性与2型 糖尿病脑梗死的关系

研究载脂蛋白E (ApoE) 基因多态性与中国东北汉族2型糖尿病合并脑梗死的关系。采用聚合酶链反应－限制性片段长度多态性（PCR-RFLP）技术,检测了208例个体的ApoE基因多态性,其中对照（CON）组69例,2型糖尿病无大血管病变（T2DM）组67例和2型糖尿病合并脑梗死（T2DMCI）组72例;同时测定了其中70例T2DMCI患者的血脂含量。CON组等位基因频率为：ε2 9.6％、ε3 82.4％、ε4 8.1％,基因型频率为：ε2ε3 13.2％、ε3ε367.6％、ε3ε416.2％;T2DM组等位基因频率为：ε2 10.5％、ε3 84.3％、ε4 5.2％,基因型频率为：ε2ε3 19.4％、ε3ε370.1％、ε3ε49％;T2DMCI组等位基因频率为：ε2 11.8％、ε3 84.7％、ε4 3.5％,基因型频率为：ε2ε3 15.2％、ε3ε375％、ε3ε44.2％。3组间等位基因和基因型频率的差异经检验无统计学意义。T2DMCI患者中各基因型之间的血浆总胆固醇 （TC）、甘油三酯 （TG）、高密度脂蛋白胆固醇（HDL-C）和低密度脂蛋白胆固醇（LDL-C）水平亦无显著性差异。在中国东北汉族人群中,未发现ApoE基因多态性与T2DMCI之间存在关联,亦未发现ApoE基因多态性与T2DMCI患者的TC、TG、 HDL-C和 LDL-C水平之间存在关联。Abstract: In order to explore the association of apolipoprotein E (ApoE) gene polymorphism with cerebral infarction in type 2 diabetic patients of Han nationality in Northeast China , the genotypes of ApoE gene were analyzed by polymerase chain reaction –restriction fragment length polymorphism (PCR-RFLP) in the 208 cases, including 69 cases in control (CON) group and 67 in type 2 diabetes mellitus (T2DM) group as well as 72 in type 2 diabetes mellitus with cerebral infarction (T2DMCI) group. Plasma lipid content in T2DMCI was also detected for 70 cases. The distribution of genotypes in ApoE gene,ε2ε3、ε3ε3 as well asε3ε4 was no significant difference in three groups (ε2ε3 : 13.2％、ε3ε3 : 67.6％、ε3ε4 : 16.2％in CON group;ε2ε3 : 19.4％、ε3ε3: : 70.1％ε3ε4 : 9％in T2DM group;ε2ε3 : 15.2％、ε3ε3 : 75％、ε3ε4 : 4.2％in T2DMCI group).The allele frequencies ofε2、ε3 andε4 were not significantly different in the three groups, either (ε2 : 9.6％、ε3 : 82.4％、ε4 : 8.1％in CON group; ε2 :10.5％、ε3 :84.3％、ε4 : 5.2％in T2DM group; ε2 :11.8％、ε3 :84.7％、ε4 : 3.5％in T2DMCI group). The levels of total cholesterol (TC), tryglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were not significantly different among the different genotypes in T2DMCI group. The study confirmed that the polymorphisms of ApoE gene are neither associated with the T2DMCI, nor with the levels of plasma lipid in T2DMCI.     

Critical role of cholesterol ester transfer protein in nicotinic acid-mediated HDL elevation in mice

Nicotinic acid is a commonly used anti-dyslipidemic agent that increases plasma levels of HDL-cholesterol and decrease triglycerides (TG), and VLDL- and LDL-cholesterol. The most well-studied effect of nicotinic acid is its ability to lower plasma free fatty acids, which has been observed in humans and many animal models. However, its ability to raise HDL in humans has not been replicated in animal models, which precludes studying the mechanism of HDL elevation. Here we studied lipid-modulating effects of nicotinic acid in mice carrying genomic DNA fragments that drive expression of various human genes in the mouse liver. Treatment with nicotinic acid reduced serum levels of HDL cholesterol in wild-type and human apolipoprotein B100 (apoB100)-transgenic mice. In contrast, nicotinic acid treatment of mice that express human cholesteryl ester transfer protein (CETP), with or without concomitant apoB100 expression, resulted in a significant increase of HDL cholesterol and reduction of TG, VLDL- and LDL-cholesterol. These data demonstrate a critical role of CETP in nicotinic acid-mediated HDL elevation, and suggest that mice carrying the human CETP gene may be useful animal models for studying the HDL-elevating effect of nicotinic acid.     

Control of apolipoprotein E secretion in the human hepatoma cell line KYN-2

Even though it is known that apolipoprotein E (apoE) is deeply involved in major age-related disorders such as atherosclerosis or Alzheimer's disease (AD), the control of cell-specific apoE expression is still poorly understood. We compared the apoE secretion as previously described in astrocytic cell17 to hepatic cell apoE secretion. We used the human hepatoma cell line KYN-2 to better delineate the characteristics of apoE secretion and to validate it with respect to the classical human hepatoma cell line HepG2. Interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) significantly inhibited, while IL-2, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were inactive on apoE secretion by KYN-2 as well as HepG2 cells. Cholesterol and 25-OH cholesterol had no effect, while forskolin exerted a significant inhibitory effect, on apoE secretion in KYN-2 cells. Our results suggest that the KYN-2 cell line represents an appropriate cell model, and in any case an alternative model to the HepG2 cell line, to study the control of apoE secretion. The response of KYN-2 cells to both cytokines and cholesterol differs from that found in astrocytoma cells, suggesting that blood variations of apoE concentrations in AD may not reflect the dysregulations taking place in the brain.     

胃转流术对2型糖尿病患者近期肠道菌群的影响

目的研究2型糖尿病患者行胃转流术前后肠道菌群是否发生变化。方法收集30例2型糖尿病患者术前和术后粪便,测肠道菌群,以25例健康志愿者的粪便为对照组。结果 2型糖尿病患者的肠杆菌较健康人增多,双歧杆菌较正常人减少,胃转流术后双歧杆菌增加,肠杆菌减少。结论 2型糖尿病患者出现肠道菌群失调,胃转流术后控制了血糖,双歧杆菌数量增加。     

Phorbol ester induces CYP2E1 in astrocytes,through a protein kinase C- and tyrosine kinase-dependent mechanism

Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide or interleukin-1beta. In the present study, phorbol-12,13-dibutyrate (PDBu) was found to induce catalytically active CYP2E1 more than fourfold in cortical glial cultures. Little induction was seen up to 12 h, and full effects only at 21-24 h of PDBu treatment. CYP2E1 expression in PDBu-treated cells was enriched in a subset of astrocytes. The protein kinase C inhibitors, staurosporine and calphostin C, and the tyrosine kinase inhibitor genistein, but not its inactive analogue daidzein, prevented the induction of CYP2E1 by PDBu. It is suggested that CYP2E1, together with interleukin-6 and ciliary neurotrophic factor, is part of a response of astrocytes to cellular stress elicited by, e.g. cerebral injury, cytokines or phorbol ester, and mediated in part through protein kinase C.     

Effect of G(-174)C polymorphism in interleukin-6 gene on cardiovascular disease in type 2 diabetes patients

Interleukin-6 (IL-6) is an important pro-inflammatory cytokine of relevance to cardiovascular diseases. The aim of this case-control study was to evaluate the association between the G(-174)C functional polymorphism in the IL-6 gene and risk of cardiovascular disease (CVD) in type 2 diabetes patients. We examined 1090 patients with T2DM and 612 controls. All subjects were genotyped for the G(-174)C polymorphism by polymerase chain reaction (PCR) and restriction analysis. There were no significant differences in the distribution of genotypes and alleles between T2DM patients and healthy controls. Significantly higher C allele frequency was observed in CVD+ patients compared to CVD- subgroup (53% vs. 32%, p < 0.0001). The odds ratio for C allele was 2.4 (95% CI 1.99–2.9, p < 0.0001) and for CC genotype 4.55 (95% CI 3.12–6.63, p < 0.000). When the distribution of G(-174)C polymorphism was compared in subgroups with different clinical phenotypes of CVD, a significant association of CC genotype with myocardial infarction was observed. Forty eight percent of patients with MI had the CC genotype compared to 22% of patients without MI (p < 0.0001). In conclusion, type 2 diabetes patients carrying the C allele of the IL-6 G(-174)C polymorphism have a significantly increased risk of CVD.     

Association of the -112A>C polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes

The -112A>C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known -3826A>G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the -112C allele and those without it. However, homeostasis model assessment for insulin resistance (p=0.0089) and HLC (p=0.012) was significantly greater in patients with the -112C allele. We did not detect an association of the -3826A>G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the -112C allele influences the susceptibility to type 2 diabetes.